Integrative in silico and biochemical analyses demonstrate direct Arl3-mediated ODA16 release from the intraflagellar transport machinery

TY - JOUR

T1 - Integrative in silico and biochemical analyses demonstrate direct Arl3-mediated ODA16 release from the intraflagellar transport machinery

AU - Wang, Jiaolong

AU - Kidmose, Rune T.

AU - Boegholm, Niels

AU - Zacharia, Nevin K.

AU - Thomsen, Mads B.

AU - Christensen, Anni

AU - Malik, Tara

AU - Lechtreck, Karl

AU - Lorentzen, Esben

N1 - Publisher Copyright: © 2025 The Authors

PY - 2025/3

Y1 - 2025/3

N2 - Outer dynein arms (ODAs) are essential for ciliary motility and are preassembled in the cytoplasm before trafficking into cilia by intraflagellar transport (IFT). ODA16 is a key adaptor protein that links ODAs to the IFT machinery via direct interaction with the IFT46 protein. However, the molecular mechanisms regulating the assembly, transport, and release of ODAs remain poorly understood. Here, we employ AlphaPulldown, an in silico screening method, to identify direct interactors of ODA16, including the dynein adaptor IDA3 and the small GTPase Arl3. We use structural modeling, biochemical, and biophysical assays on Chlamydomonas and human proteins to elucidate the interactions and regulatory mechanisms governing the IFT of ODAs. We identify a conserved N-terminal motif in Chlamydomonas IFT46 that mediates its binding to one side of the ODA16 structure. Biochemical dissection reveals that IDA3 and Arl3 bind to the same surface of ODA16 (the C-terminal β-propeller face), which is opposite to the IFT46 binding site, enabling them to dissociate ODA16 from IFT46, likely through an allosteric mechanism. Our findings provide mechanistic insights into the concerted actions of IFT and adaptor proteins in ODA transport and regulation.

AB - Outer dynein arms (ODAs) are essential for ciliary motility and are preassembled in the cytoplasm before trafficking into cilia by intraflagellar transport (IFT). ODA16 is a key adaptor protein that links ODAs to the IFT machinery via direct interaction with the IFT46 protein. However, the molecular mechanisms regulating the assembly, transport, and release of ODAs remain poorly understood. Here, we employ AlphaPulldown, an in silico screening method, to identify direct interactors of ODA16, including the dynein adaptor IDA3 and the small GTPase Arl3. We use structural modeling, biochemical, and biophysical assays on Chlamydomonas and human proteins to elucidate the interactions and regulatory mechanisms governing the IFT of ODAs. We identify a conserved N-terminal motif in Chlamydomonas IFT46 that mediates its binding to one side of the ODA16 structure. Biochemical dissection reveals that IDA3 and Arl3 bind to the same surface of ODA16 (the C-terminal β-propeller face), which is opposite to the IFT46 binding site, enabling them to dissociate ODA16 from IFT46, likely through an allosteric mechanism. Our findings provide mechanistic insights into the concerted actions of IFT and adaptor proteins in ODA transport and regulation.

KW - Arl3

KW - cilium

KW - FAP20

KW - flagellum

KW - IDA3

KW - IFT46

KW - intraflagellar transport

KW - ODA16

KW - ODA8

KW - outer dynein arms

UR - http://www.scopus.com/inward/record.url?scp=85217911412&partnerID=8YFLogxK

U2 - 10.1016/j.jbc.2025.108237

DO - 10.1016/j.jbc.2025.108237

M3 - Journal article

C2 - 39880089

AN - SCOPUS:85217911412

SN - 0021-9258

VL - 301

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 3

M1 - 108237

ER -